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P F Bray

Publications and source records attributed to P F Bray.

At least 37 records · Page 2Linked to original sources
PubMed1999

Integrin polymorphisms as risk factors for thrombosis.

The study of integrin polymorphisms as risk factors for thrombosis is in its infancy. Like many other purported genetic risk factors, controversies abound, and it will take much work to shift through the information and identify those individuals for whom these genetic variations are true risks for thrombosis. There is a clear need for well-designed, large, prospective, genetic epidemiologic studies. Large clinical trials should be helpful in this regard, and genotyping patients in these studies will be the only way to correlate response to therapy with genetic factors (pharmacogenetics). Hand-in-hand with such clinical investigations, in vitro studies of the functional consequences of these inherited traits should provide a sound rationale for future interventions that will ultimately be beneficial to patients.

Animals↗
PubMed1998

R to Q amino acid substitution in the GFFKR sequence of the cytoplasmic domain of the integrin IIb subunit in a patient with a Glanzmann's thrombasthenia-like syndrome.

The integrin IIbbeta3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and beta3. We now report the molecular analysis of IIbbeta3 from a patient with a Glanzmann's thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbbeta3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the beta3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type beta3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and beta3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary-transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

Amino Acid Substitution↗
PubMed1998

Human integrin beta3 gene expression: evidence for a megakaryocytic cell-specific cis-acting element.

The human integrin beta3 participates in a wide range of adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for beta3 expression, a number of upstream regulatory regions in the beta3 gene were identified. (1) The three regions from -1159 to -584, -290 to -146, and -126 to -115 demonstrated positive, negative, and negative activity, respectively. (2) The region from -115 to +29 of the beta3 gene was sufficient for cell-specific activity. Deletion of the sequence from -115 to -89 produced a 6- to 40-fold reduction in reporter gene activity in beta3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and 2.7-fold reduction, respectively, in beta3-expressing endothelial and melanoma cell lines, and 1.3- and 2. 8-fold reduction, respectively, in non-beta3-expressing Chinese hamster ovary and 293 cell lines. This sequence also bound nuclear proteins in a cell-specific manner in electrophoretic mobility shift assays. Mutational analysis indicated that the sequence GAGGGG (positions -113 to -108) is a megakaryocytic cell line-specific cis-acting element. (3) The region from -89 to +29 promoted lower activity in all cell lines. We also provide evidence that a CCCACCC sequence at position -70 has transcriptional activity, most likely through the Sp1 transcription factor. These data supply the first detailed map of the transcriptional regulatory elements of the 5' region of the beta3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment beta3 gene expression.

Animals↗
PubMed1998

Priming of platelet alphaIIbbeta3 by oxidants is associated with tyrosine phosphorylation of beta3.

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.

Humans↗
PubMed1998

Platelet glycoprotein receptor IIIa polymorphism P1A2 and ischemic stroke risk: the Stroke Prevention in Young Women Study.

BACKGROUND AND PURPOSE: Platelet glycoprotein IIb/IIa (GpIIb-IIIa), a membrane receptor for fibrinogen and von Willebrand factor, has been implicated in the pathogenesis of acute coronary syndromes but has not been previously investigated in relation to stroke in young adults. METHODS: We used a population-based case-control design to examine the association of the GpIIIa polymorphism P1A2 with stroke in young women. Subjects were 65 cerebral infarction cases (18 patients with and 47 without an identified probable etiology) 15 to 44 years of age from the Baltimore-Washington region and 122 controls frequency matched by age from the same geographic area. A face-to-face interview for vascular disease risk factors and a blood sample for the P1A2 allele and serum cholesterol were obtained from each participant. Logistic regression was used to estimate the odds ratio for one or more P1A2 alleles after adjustment for other risk factors. RESULTS: Among cases and controls, the prevalence rates of one or more P1A2 alleles were 21% and 22% among blacks and 36% and 28% among whites, respectively. This genotype was significantly associated with hypertension only in black control subjects but otherwise not with any of the established vascular risk factors. The adjusted odds ratio for cerebral infarction of one or more P1A2 alleles was 1.1 (confidence interval [CI], 0.6 to 2.3) overall, 0.5 (CI, 0.1 to 7.1) among blacks, and 1.4 (CI, 0.5 to 3.7) among whites. For the cases with an identified probable etiology, the corresponding odds ratios were 3.0 (CI, 0.9 to 10.4) overall, 0.7 (CI, 0.1 to 7.1) among blacks, and 12.8 (CI, 1.2 to 135.0) among whites. CONCLUSIONS: No association was found between the P1A2 polymorphism of GpIIIa and young women with stroke. However, subgroup analyses showed that the P1A2 polymorphism of GpIIIa appeared to be associated with stroke risk among white women, particularly those with a clinically identified probable etiology for their stroke. Further work with an emphasis on stroke subtypes and with multiracial populations is warranted.

Adolescent↗
PubMed1997

The human integrin beta3 gene is 63 kb and contains a 5'-UTR sequence regulating expression.

The human blood platelet fibrinogen receptor, integrin alphaIIbbeta3 (glycoprotein IIb-IIIa) is an archetypal member of the integrin family of adhesive molecules and is the only integrin encoded by genes physically linked in the genome. Because studies on the normal and abnormal expression of any gene require a thorough understanding of its organization, the initial goals of the current study were to determine the size and complete the genomic organization for the beta3 gene. We now report the isolation of the entire beta3 gene in a single P1 plasmid and for the first time have linked the first and second exons on a contiguous fragment of DNA. Using pulsed-field gel analysis, we determined the full size of the beta3 gene to be 63 kb and show a large (16.7 kb) first intron; based on this information, we propose a uniform numbering system for the beta3 exons. We have completed the 5' genomic structure and generated a long-range restriction map. The promoter and the 5' end of the first intron were found to have approximately 50% sequence identity with a region of the avian beta3 gene known to possess functional transcriptional activity. Analysis of three different homologous regions led to the identification of a sequence in the 5'-UTR of the human gene, CCGCGGGAGG, which shares 90% identity with the avian gene and which bound nuclear proteins in DNaseI and electrophoretic mobility shift assay studies. Mutating this sequence caused a 2.6-fold reduction in reporter gene activity. In these studies we have (1) determined the full length and 5' organization of the beta3 gene, (2) identified a large region of homology between the 5' regions of the avian and human genes, and (3) identified a sequence in the 5'-UTR that augments gene expression. Knowing the genomic structure of beta3 has permitted the uncovering of new mechanisms of mutagenesis causing Glanzmann thrombasthenia (Jin et al, J Clin Invest 98:1745, 1996), and our findings will be valuable for such genetic analyses as well as for studies on the transcriptional regulation of beta3 and other integrin genes.

Antigens, CD↗
PubMed1997

Gender differences in platelet GPIIb-IIIa activation.

Gender differences in the development of thrombotic diseases have been described in numerous clinical settings. Enhanced platelet reactivity in both sexes is associated with the development of vascular thromboses. Because activation of platelet GPIIb-IIIa receptors is a central event in thrombus formation, we examined GPIIb-IIIa function in normal male and female volunteers. Using flow cytometry, we quantitated gender differences in the number of binding sites for FITC-labeled fibrinogen (FITC-FGN) and FITC-labeled PAC-1 antibody (FITC-PAC-1). Washed platelets were incubated with either FITC-FGN or FITC-PAC-1 and activated with either ADP or thrombin receptor activating peptide (TRAP) prior to cytometric acquisition of data. The dissociation constant for FITC-FGN was the same in both sexes (approx. 1.6 x 10(-7)M), however, the number of GPIIb-IIIa receptors per platelet capable of binding fibrinogen was significantly greater in women than men in response to 2 microM ADP (16,319 +/- 1871 vs 9669 +/- 1994, p = 0.02), 20 microM ADP (39,951 +/- 4711 vs 25,948 +/- 4953, p = 0.05) and 50 microM TRAP (39,236 +/- 3965 vs 21,848 +/- 4159, p = 0.007). Similarly, the number of GPIIb-IIIa receptors capable of binding PAC-1 in response to ADP and TRAP was 50% to 80% greater in women than men. Binding experiments using specific anti-GPIIb-IIIa monoclonal antibodies (P2 and 10E5), as well as quantitative Western blotting experiments, showed no gender difference in the total number of GPIIb-IIIa molecules expressed. Analysis of data from female subgroups demonstrated an association of GPIIb-IIIa reactivity with menstrual phase. We conclude that GPIIb-IIIa receptors on platelets from premenopausal women are more "activatable" than those on platelets from young men. Variations in the serum concentrations of estrogens and/or progestins may modulate GPIIb-IIIa function.

Adult↗
PubMed1996

Effects of integrelin, a platelet glycoprotein IIb/IIIa receptor antagonist, in unstable angina. A randomized multicenter trial.

BACKGROUND: Although aspirin is beneficial in patients with unstable angina, it is a relatively weak inhibitor of platelet aggregation. The effect of Integrelin, which inhibits the platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa, on the frequency and duration of Holter ischemia was evaluated in 227 patients with unstable angina. METHODS AND RESULTS: Patients received intravenous heparin and standard ischemic therapy and were randomized to receive oral aspirin and placebo Integrelin; placebo aspirin and low-dose Integrelin. 45 micrograms/kg bolus followed by a 0.5 microgram.kg-1. min-1 continuous infusion; or placebo aspirin and high-dose Integrelin, 90 micrograms/kg bolus followed by a 1.0-microgram.kg-1, min-1 constant infusion. Study drug was continued for 24 to 72 hours, and Holter monitoring was performed. Patients randomized to high-dose Integrelin experienced 0.24 +/- 0.11 ischemic episodes (mean +/- SEM) on Holter lasting 8.41 +/- 5.29 minutes over 24 hours of study drug infusion. Patients randomized to aspirin experienced a greater number (1.0 +/- 0.33, P < .05) and longer duration (26.2 +/- 9.8 minutes, P = .01) of ischemic episodes than the high-dose Integrelin group. There was no evidence of rebound ischemia after withdrawal of study drug. In 46 patients, platelet aggregation was rapidly inhibited by Integrelin in a dose-dependent fashion. The number of clinical events was small, and there were no bleeding differences in the three treatment arms. CONCLUSIONS: Intravenous Integrelin is well tolerated, is a potent reversible inhibitor of platelet aggregation, and added to full-dose heparin reduces the number and duration of Holter ischemic events in patients with unstable angina compared with aspirin.

Adult↗
PubMed1996

Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17.

Adult↗
PubMed1996

A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis.

BACKGROUND: Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggregation. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, PlA2, of the gene encoding glycoprotein IIIa in kindreds with a high prevalence of premature myocardial infarction. METHODS: To investigate the relation between the PlA2 polymorphism and acute coronary syndromes, we conducted a case-control study of 71 case patients with myocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the PlA genotype: reverse dot blot hybridization and allele-specific restriction digestion. RESULTS: The prevalence of PlA2 was 2.1 times higher among the case patients than among the controls (39.4 percent vs. 19.1 percent, P=0.01). In a subgroup of patients whose disease began before the age of 60 years, the prevalence of PlA2 was 50 percent, a value that was 3.6 times that among control subjects under 60 years of age (13.9 percent, P=0.002). Among subjects with the PlA2 polymorphism, the odds ratio for having a coronary event was 2.8 (95 percent confidence interval, 1.2 to 6.4). In the patients less than 60 years of age at the onset of disease, the odds ratio was 6.2 (95 percent confidence interval, 1.8 to 22.4). CONCLUSIONS: We observed a strong association between the PlA2 polymorphism of the glycoprotein IIIa gene and acute coronary thrombosis, and this association was strongest in patients who had had coronary events before the age of 60 years.

Acute Disease↗
PubMed1996

Refining the treatment of women with unstable angina--a randomized, double-blind, comparative safety and efficacy evaluation of Integrelin versus aspirin in the management of unstable angina.

BACKGROUND: Although women typically develop coronary artery disease several years after men, once they have symptomatic disease their thromboembolic complications are worse than in men. The mechanism mediating this gender difference in outcome after thromboembolic events is unknown. We previously studied platelet functions in siblings from patients with premature coronary artery disease. We observed that platelets from women are responsive than their male counterparts. In particular, platelets from women stimulated ex vivo with various agonists bind more fibrinogen molecules than platelets from men. HYPOTHESIS: We hypothesized that in patients with acute coronary events, the control of platelet activity might require stronger antagonists in women than in men. METHODS: To test this hypothesis, we investigated retrospectively the results of a trial on Integrelin in unstable angina. RESULTS: We report that platelet aggregation and Holter-detected ischemic episodes are significantly reduced in women with unstable angina treated with the specific GPIIb-IIIa inhibitor, Integrelin, compared with the standard platelet inhibitor aspirin. In contrast, both platelet aggregation and Holter-detected ischemic events are well controlled in men with unstable angina treated with standard therapy including aspirin. CONCLUSION: Integrelin does provide protection in men, but, in contrast with women, not beyond what can be achieved with aspirin. Our data are consistent with the concept that the platelets from women require stronger and more specific inhibitors to limit their activity, and that platelets may play a more important role in women with acute coronary syndromes than in men. Most important, specific GPIIb-IIIa inhibitors may represent a therapeutic option which provides as much suppression of ischemic events in women as they do in men with coronary artery disease.

Aged↗
PubMed1996

The role of HLA antibodies in neonatal thrombocytopenia: a prospective study.

The role of HLA antibodies in neonatal alloimmune thrombocytopenia is controversial. We prospectively studied the sera of obstetric patients at delivery for HLA antibodies and correlated their presence with umbilical cord blood platelet counts. We studied 493 births at The Johns Hopkins Hospital comprising of 357 African American, 115 Caucasian, and 21 babies of other racial groups. One hundred and thirty nine mothers had HLA antibodies. Of these HLA alloimmunized mothers, only ten infants had platelet counts of 150,000/ microL or less. Three hundred and eight mothers with no detectable antibodies gave birth to 27 infants with platelet counts of 150,000/microL or less. Yates corrected Chi square analysis showed no significant relationship between maternal HLA alloimmunization and baby platelet count (p = 0.709). Only 8 of sixty cord sera from babies of HLA alloimmunized mothers were positive for HLA antibodies. The HLA cord blood antibody results were then correlated with the neonatal platelet counts. The Fisher's exact test showed no significant relationship between the presence of HLA antibodies in cord blood samples and neonatal platelet counts (p = 0.232). Although one third (31%) of mothers have HLA antibodies, neonatal thrombocytopenia is rarely associated with this finding. However, HLA antibodies can cross the placenta, and in these unusual cases, may be associated with a higher risk of neonatal thrombocytopenia.

Female↗
PubMed1995

Localization of a binding site for phosphatidylinositol 4,5-bisphosphate on human profilin.

Profilin is a small 12-15-kDa actin-binding protein, which in eukaryotic organisms is ubiquitous and necessary for normal cell growth and function. Although profilin's interactions with its three known ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP2), and poly-L-proline (PLP)) have been well characterized in vitro, its precise role in cells remains largely unknown. By binding to clusters of PIP2, profilin is able to inhibit the hydrolysis of PIP2 by phospholipase C gamma 1 (PLC gamma 1). This ability is the result of profilin's affinity for PIP2, but the specific residues of profilin's amino acid sequence involved in the binding of PIP2 are not known. Using site-directed mutagenesis, we sought to localize regions of profilin important for this interaction by generating the following mutants of human profilin (named according to the wild-type amino acid altered, its position, and the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53I, R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of L10R, all of the mutants were successfully expressed in Escherichia coli and purified by affinity chromatography on PLP-Sepharose. Only Y6F and K25Q demonstrated moderately less stringent binding to PLP, indicating that most of the mutations did not induce marked alterations of profilin's structure. When tested for their relative abilities to inhibit the hydrolysis of PIP2 by PLC gamma 1, most of the mutants were indistinguishable from wild-type profilin. Exceptions included D8A, which demonstrated increased inhibition of PLC gamma 1, and R88L, which demonstrated decreased inhibition of PLC gamma 1. To assess the importance of the region surrounding residue 88 of human profilin, three synthetic decapeptides selected to correspond to non-overlapping stretches of the human profilin sequence were tested for their abilities to inhibit PLC gamma 1. We found that only te decapeptide that matched the peptide stretch centered around residue 88 was able to inhibit PLC gamma 1 activity substantially and was able to do so at nearly wild-type profilin levels. Taken together with the finding that mutating residue 88 resulted in decreased inhibition of PLC gamma 1 activity, these data provide strong evidence that this region of human profilin represents an important binding site for PIP2.

Actins↗